Herpetology Notes, volume 8: 347-356 (2015) (published online on 16 June 2015) Reproductive biology of Ambystoma salamanders in the southeastern United States Brad M. Glorioso1,*, J. Hardin Waddle1 and Jeromi Hefner2 Abstract. Reproductive aspects of Ambystoma salamanders were investigated at sites in Louisiana (2010–12) and Mississippi (2013). Three species occurred at the Louisiana site, Spotted Salamander (A. maculatum), Marbled Salamander (A. opacum), and Mole Salamander (A. talpoideum), whereas only Spotted Salamanders were studied at the Mississippi site. A total of 162 and 71 egg masses of Spotted Salamanders were examined at the Louisiana and Mississippi sites, respectively. Significantly more Spotted Salamander eggs per egg mass were observed at the Mississippi site (x̄ = 78.2) than the Louisiana site (x̄ = 53.8; P < 0.001). The mean snout–vent length of female Spotted Salamanders at the Mississippi site (82.9 mm) was significantly larger than the Louisiana site (76.1 mm; P < 0.001). Opaque Spotted Salamander egg masses were not found at the Mississippi site, but accounted for 11% of examined egg masses at the Louisiana site. The mean number of eggs per egg mass at the Louisiana site did not differ between opaque (47.3) and clear (54.6) egg masses (P = 0.21). A total of 47 egg masses of the Mole Salamander were examined, with a mean number of 6.7 embryos per mass. Twenty-three individual nests of the Marbled Salamander were found either under or in decaying logs in the dry pond basins. There was no difference between the mean numbers of eggs per mass of attended nests (93.0) versus those that were discovered unattended (86.6; P = 0.67). Females tended to place their nests at intermediate heights within the pond basin. Keywords. Ambystoma maculatum, A. opacum, A. talpoideum, reproduction, clutch size, nest site selection, Louisiana, Mississippi Introduction A. maculatum (Shaw, 1802) and the Mole Salamander, A. talpoideum (Holbrook, 1838), lay their eggs in winter Ambystoma is a genus of North American salamanders or early spring in ponds that have already filled with in the family Ambystomatidae, known collectively as water (Fig. 1). However, a few species, including the the mole salamanders. Though there are some neotenic Marbled Salamander, A. opacum (Gravenhorst, 1807), examples, most Ambystoma are terrestrial, spending the lay eggs in the fall in a constructed nest in the dry pond majority of their time in underground burrows. They basin (or at the periphery of a reduced pond) before are nocturnal and most typically observed during their it fills (Fig. 1; Dunn, 1917; Petranka and Petranka, reproductive season when they emerge from underground 1981a). at night and migrate to their breeding ponds. Most A single female Spotted Salamander typically lays 2–4 terrestrial Ambystoma, such as the Spotted Salamander, masses of eggs per year. The large, firm globular egg masses, each typically containing 1–250 eggs, are laid in relatively shallow areas of the pond either attached to vegetation or resting on the pond bottom (Bishop, 1941; Shoop, 1974). Mole Salamanders lay eggs singly in 1 U.S. Geological Survey, National Wetlands Research Center, Atlantic Coastal Plain populations, but in Gulf Coastal 700 Cajundome Boulevard, Lafayette, Louisiana 70506, Plain populations a single female lays many small USA. delicate clusters of eggs, usually attached to vegetation 2 Five Rivers Services, LLC, National Wetlands Research Center, 700 Cajundome Boulevard, Lafayette, Louisiana (Semlitsch and Walls, 1990; Trauth, 2005). Although it 70506, USA. is very difficult to determine the female responsible for * Corresponding author: E-mail: [email protected] a particular egg mass in Spotted and Mole Salamanders, 348 Brad M. Glorioso et al. The Marbled Salamander is unique in that it is the only Ambystoma species that exhibits parental care in the form of nest-guarding (Nussbaum, 1985, 1987). Female nest site selection may be influenced by factors such as microsite elevation within the pond basin (Petranka and Petranka, 1981a; Jackson et al., 1989), pond hydrology (Wojnowski, 2000), cover availability (Petranka and Petranka, 1981b; Figiel and Semlitsch, 1995), and soil moisture (Figiel and Semlitsch, 1995; but see Marangio and Anderson, 1977). The length of time that females brood their clutch is variable, but females may leave the clutch before inundation (McAtee, 1933; Jackson et al., 1989; Petranka, 1990). Although the mechanism is not fully known, it appears nest brooding in Marbled Salamanders enhances embryonic survival, perhaps by defending against predators, fungal invasion, and desiccation (Petranka and Petranka, 1981b; Jackson et al., 1989; Petranka, 1998; Croshaw and Scott, 2005). We report clutch and egg mass data and other aspects of reproduction of three species of Ambystoma salamanders. Our data span two and a half breeding seasons at a site in Kisatchie National Forest in Louisiana and one breeding season at a site along the Natchez Trace National Parkway in Mississippi. Though limited in scope, the data reported herein contributes to the reproductive knowledge of these species. Materials and Methods We conducted the work reported herein primarily in the Kisatchie District of Kisatchie National Forest in Natchitoches Parish, Louisiana, USA, but also along the Natchez Trace National Parkway in Hinds County, Mississippi, USA (Fig. 2). In Kisatchie, we began our examination of the ambystomatid salamander community in the fall of 2010 at two nearby ponds and associated wetland areas within the Kisatchie Bayou Recreation Area (Fig. 3). The study area in Kisatchie has Figure 1. The three salamander species and their eggs that are discussed in this paper. (A) Spotted Salamander (Ambystoma not been logged in over 100 years and is forested with maculatum), clear egg mass (inset lower left), and opaque egg large hardwoods and pines. The study area is bordered th mass (inset lower right). (B) Mole Salamander (A. talpoideum) on the west by Kisatchie Bayou (a 7 order stream), on in water, egg mass (inset lower right). (C) Marbled Salamander the east by steep hills, and to the north and northeast by (A. opacum) guarding her clutch that was laid under a log. a large clearcut (Fig. 3). Photos in (A) and (C) by Brad M. Glorioso. Photos in (B) by Mole Pond at the Kisatchie site is a large, relatively Jeromi Hefner. deep (~2 m at its deepest) ephemeral pond with ample leaf litter and coarse woody debris. Several large pine (Pinus sp.) and gum (Nyssa sp.) trees are at the edges the entire clutch (30–200 eggs) of a female Marbled and shallower portions of the pond, with buttonbush Salamander is laid at once, typically under a log or (Cephalanthus occidentalis) occupying the deepest other cover, and attended by the female for some time portions. Ox Pond is a deeply cut old oxbow of Kisatchie thereafter (Petranka, 1998). Bayou, and is filled with bald cypress (Taxodium Reproductive biology of Ambystoma salamanders in the southeastern United States 349 injecting them with a Passive Integrated Transponder (PIT) tag. At the Natchez Trace site, we used a drift fence that encircled the breeding pond to capture Spotted Salamanders migrating into the pond in 2013. We sexed and measured each salamander for SVL, TL, and body mass before injecting them with Visible Implant Elastomer (VIE) batch marks as part of a larger study on salamander migration. We discovered egg clutches of the Marbled Salamander in Kisatchie by turning logs in the bottom of Mole and Ox ponds as well as Ditch. When a clutch was found attended by a female, we processed the female as stated above and placed the entire egg clutch into a pour-boat. With the pour-boat angled downwards at the clutch site, we counted the eggs as we carefully returned them singly or in small groups back to the oviposition site. We then returned the female to her clutch, and carefully replaced the log. We noted the location with colored Figure 2. Location of the study areas within Louisiana and pin flags marked with the nest number we assigned. On Mississippi, USA. distichum) throughout the pond basin. Whereas Mole Pond typically fills with rainwater and runoff from the nearby hills, Ox Pond typically fills from flooding of the bayou. The third major breeding area at the Kisatchie site is Ditch, a relatively shallow (depth < 1 m) sinuous intermittently flooded waterway that is connected to Ox Pond and travels through the middle of the study area. Other depressions in the study area are insignificant to Ambystoma reproduction. The pond at the Natchez Trace site is man-made, rectangular in shape, and surrounded by a low berm. Oak (Quercus sp.) dominates the surrounding forest. The water in the breeding pond is permanent, which is unusual with respect to typical breeding ponds used by Ambystoma, but is suitable for breeding because it lacks fish predators. The pond is relatively small compared to the Kisatchie ponds, with moderate depth (> 1 m at its deepest), and is covered with duckweed (Lemna sp.) on the surface, with a very soft bottom. We searched for ambystomatid salamanders at the Kisatchie site primarily by turning logs both within Figure 3. Elevation of the study site showing the locations of the dry pond basins and the associated upland areas. the primary Ambystoma breeding sites in Kisatchie National We also used unbaited minnow traps to capture adult Forest and their relative position to Kisatchie Bayou to the salamanders within the subsequently flooded ponds. We west and the hills to the east (top) and aerial imagery of the sexed and measured each salamander for snout–vent study site showing the clearcut to the northeast (bottom). The length (SVL), total length (TL), and body mass before scale applies to both images. 350 Brad M. Glorioso et al. 1 Table 1.